Smokeless tobacco consumed by 12 million Americans is causatively associated with cancer of the oral cavity and pharynx. In ongoing work with supercritical fluid extraction, we have demonstrated that there is more of the powerfull carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK) present in smokeless tobacco than had been assayed so far by conventional analytical studies. Additional amounts of this potent carcinogen are bound to macromolecules in the tobacco. This bound NNK, while not extractable by conventional methods, is conceivably released from tobacco during chewing. One of the primary aims of this project is to determine the bioavailability of bound NNK and other tobacco carcinogens to the smokeless tobacco user, the mechanism of its release, and the nature of its binding in tobacco. More than 200 million people in India and in other Asian countries practice chewing of betel quid, mostly containing tobacco which is causally associated with cancer of the oral cavity and esophagus. The presence of N-nitrosation products of arecoline, the major alkaloid of betel quid, in saliva of betel quid chewers has first ben demonstrated by us. One of these compounds, namely, 3-(methylnitrosamino) propionitrile (MNPN) is a powerful carcinogen in rats, yet it failed to induce tumors of the laboratory animals. The presence of other compounds is probably needed for the carcinogenic potency of the betel quid in the oral cavity and esophagus. Since there exists only very limited knowledge as to the chemical composition and biological activity of betel quid, we plan to analyze it for its major, bioavailable components and evaluate the newly identified agents for their irritating potential and tumorigenicity.